淫羊藿苷对MC3T3-E1成骨前体细胞的成骨诱导作用及意义

Osteoinductivity of icariin on MC3T3-E1 cells

目的观察淫羊藿苷对MC3T3-E1成骨前体细胞的细胞毒性及成骨诱导作用,为其用于骨组织再生提供依据。方法将MC3T3-E1成骨前体细胞随机分为观察组、阴性对照组及阳性对照组。观察组分别加入终浓度为1×10^(-6)、10^(-5)、1×10^(-4)、1×10^(-3)、1×10^(-2)mmol/L的淫羊藿苷溶液100μL,阴性对照组及阳性对照组分别加入等体积含DMSO的培养基及骨形态发生蛋白2(rh BMP-2)。采用MTT法检测各组培养第3、7天的细胞增殖率,死/活细胞荧光染色法检测细胞存活率,茜素红钙结节染色观察细胞钙化结节形成情况,细胞超声裂解法检测碱性磷酸酶(ALP)活性、钙含量及骨钙素表达。结果培养第3天,加入1×10^(-2)mmol/L淫羊藿苷溶液的观察组细胞增殖率明显低于阴性对照组及阳性对照组(P均<0.05)。培养第7天,加入1×10^(-3)、1×10^(-2)mmol/L淫羊藿苷溶液的观察组细胞增殖率明显低于阴性对照组及阳性对照组(P均<0.05)。加入1×10^(-3)mmol/L淫羊藿苷溶液的观察组细胞存活率明显低于阳性对照组,加入1×10^(-2)mmol/L淫羊藿苷溶液的观察组细胞存活率明显低于阴性对照组及阳性对照组(P均<0.05)。阴性对照组未见明显红染钙化结节,阳性对照组有明显的钙化结节,观察组随着加入淫羊藿苷浓度的升高红染钙化结节面积逐渐增大。加入不同浓度淫羊藿苷溶液的观察组细胞ALP活性、骨钙素表达均高于阴性对照组(P均<0.05)。加入1×10^(-4)、1×10^(-3)、1×10^(-2)mmol/L淫羊藿苷的观察组细胞钙含量均高于阴性对照组,加入1×10^(-6)、1×10^(-5)mmol/L淫羊藿苷的观察组细胞钙含量均低于阳性对照组(P均<0.05)。结论 1×10^(-4)mmol/L淫羊藿苷溶液对MC3T3-E1成骨前体细胞的细胞毒性较低,其成骨诱导作用与rh BMP-2接近,可作为一种骨诱导生长因子替代剂联合骨修复材料用于促进骨组织再生。

Objective To evaluate cytotoxicity and osteoinductivity of icarrin on MC3 T3-E1 osteoblast precursor cells,which provides basis for bone tissue regeneration. Methods The MC3 T3-E1 cells were randomly divided into the observation group,the negative control group,and the positive group. The cells in the observation group were cultured with 100μL of 1 × 10^-6,1 × 10^-5,1 × 10^-4,1 × 10^-3,and 1 × 10^-2 mmol/L icarrin,respectively; cells in the negative control group were added with the same volume of DMSO and cells in the positive group with the same volume of rh BMP-2. Subsequently,the proliferation rate on the 3 rd and 7 th day was tested with MTT method. The cell survival rate was determined by dead/living cell fluorescence detection. The calcium nodules were observed by Alizarin red calcium nodules stain. The calcium content,alkaline phosphatase（ ALP）,and osteocalcin were tested by cellular ultrasonic cracking. Results The cell proliferation rate in the observation group with 1 × 10^-2 mmol/L icariin was lower than that in the positive and negative groups on the 3 rd day（ both P〈0. 05）,and the cell proliferation rate in the observation group with 1 × 10^-2 mmol/L and 1 × 10^-3 mmol/L icariin was lower than that in the positive and negative groups on the 7 th day（ both P〈0. 05）. The cell survival rate in the observation group with 1 × 10^-3 mmol/L icariin was lower than that in the positive group（ P〈0. 05）. The cell survival rate in the observation group with 1 × 10^-2 mmol/L icariin was lower than that in the positive and negative groups（ both P〈0. 05）. No calcium nodules formed in the negative group and a large area of calcium nodules was found in the positive group. In the observation group,the calcium nodule gradually increased with the increasing concentrations of icariin. The ALP activity and osteocalcin in the observation group were higher than those in the negative group（ all P〈0. 05）.The Ca content in each of the observation group with 1 × 10^-4,1 × 10^-3,and 1 × 10^-2 mmol/L icariin was higher than that in the negative group,and the Ca content in each of the observation group with 1 × 10^-6,and 1 × 10^-5 mmol/L icariin was lower than that of the positive group（ all P〈0. 05）. Conclusion 10^-4 mmol/L icariin,with lower cytotoxicity and osteoinductivity,which is similar to rh BMP-2,can be used as an alternative agent of growth factor combined with bone repair materials to promote the bone tissue regeneration.